Whole genome sequencing in adults with clinical hallmarks of hypophosphatasia negative for ALPL variants

Background Hypophosphatasia (HPP) is a rare disease caused by deficient activity of tissue-nonspecific alkaline phosphatase (ALP), encoded by the ALPL gene. The primary objective was to explore novel ALPL variants by whole genome sequencing (WGS) in patients with HPP who previously tested negative by standard methods for ALPL variants. The secondary objective was to search for genes beyond ALPL that may reduce ALP activity or contribute to HPP symptoms. Methods and results WGS was performed in 16 patients clinically diagnosed with HPP who had ALP activity below the normal range and tested negative for ALPL variants. Genetic variants in ALPL and genes possibly associated with low ALP activity or phenotypic overlap with HPP were assessed. All 16 patients had ALP activity below the normal range. WGS did not identify any novel disease-causing ALPL variants. Positive findings for other gene variants were identified in 4 patients: 1 patient presented with variants in COL1A1, NLRP12, and SCN9A, coding for collagen, type, I alpha-1 chain, nod-like receptor pyrin domain containing 12, and sodium voltage-gated channel alpha subunit 9, respectively; 1 presented with a heterozygous, likely pathogenic variant in P3H1 coding for prolyl 3 hydroxylase 1; 1 presented with a heterozygous pathogenic variant in SGCE, coding for sarcoglycan epsilon; and 1 presented with a heterozygous variant of uncertain significance in VDR, encoding vitamin D receptor. Conclusion Genomic analysis did not identify novel ALPL variants or a pattern of disease-causing variants in genes other than ALPL to explain the HPP phenotype in these patients. Registration Clinicaltrials.gov identifier: NCT04925804.


Introduction
Hypophosphatasia (HPP) is a rare, metabolic disease caused by deficient activity of the enzyme tissue-nonspecific alkaline phosphatase (ALP), encoded by the ALPL gene [1].Low ALP activity may result in accumulation of its substrates, including inorganic pyrophosphate (PPi) and pyridoxal 5ʹ-phosphate (PLP).The accumulation of PPi, a potent inhibitor of bone mineralization, can cause defective skeletal mineralization, while impaired dephosphorylation

ALP Alkaline Phosphatase CNV Copy Number Variants ExAC
Exome Aggregation Consortium GWAS Genome-Wide Association Study HPP Hypophosphatasia KEGG Kyoto Encyclopedia of Genes and Genomes MDS Myoclonus Dystonia Syndrome OMIM Online Mendelian Inheritance in Man of PLP into pyridoxal can result in vitamin B 6 -responsive seizures [1,2].While the clinical spectrum of HPP is extremely broad across different age groups, it is often associated with severe systemic morbidity, particularly due to musculoskeletal deficits (e.g., fractures, muscle weakness) and pain, in patients manifesting the disease later in life [3].
Accurate, timely diagnosis of HPP is important for appropriate disease management [1].A clinical diagnosis of HPP is based on identification of characteristic manifestations [1].Differential diagnosis involves the exclusion of other pathologic conditions associated with phenotypic overlap and/or low ALP activity [1].Sequencing of the ALPL coding region can be used to confirm a diagnosis of HPP [1] and identifies variants in approximately 95% of patients with the disease [4].To date, more than 400 variants of the ALPL gene, predominantly missense variants, have been identified [5], indicating substantial allelic heterogenicity.However, standard sequencing techniques may fail to detect ALPL variants in some patients because of the presence of new variants not yet classified as pathogenic, variants within introns or within the regulatory sequences of ALPL, large deletions/duplications not typically assessed by standard methods, or variants in other genes involved in the regulation of ALPL [6,7].
The primary objective of this study was to identify novel ALPL variants by whole gene sequencing (WGS) in patients with HPP who previously tested negative by standard methods for ALPL variants.The secondary objective was to search for genes beyond ALPL that may reduce ALP activity or contribute to HPP symptoms in these patients.

Objectives
The primary study objective was to compare the results of WGS with those of standard sequencing carried out for detection of ALPL variants.The secondary objective was to search for gene variants within or beyond ALPL that could result in low ALP activity.

Patients
This prospective, observational cohort study (NCT04925804) was conducted between June 2 and December 2, 2021, at a single HPP expert center at University of Würzburg, Germany.Eligible patients had a clinical diagnosis of HPP based on age-and sex-adjusted low serum ALP activity and clinical symptoms.ALP activity was determined on 2 occasions, at least 1 month apart, using a commercially available assay.Patients were required to have an available sequencing report indicating no pathogenic variants, no likely pathogenic variants, or no variants of uncertain significance (VUS) in ALPL.Patients were excluded if they had other potential causes of low ALP, including celiac disease, clofibrate therapy, cleidocranial dysplasia, Cushing's syndrome, hypothyroidism, massive blood transfusion, milk-alkali syndrome, multiple myeloma, osteogenesis imperfecta type II, pernicious or profound anemia, starvation, vitamin C deficiency, vitamin D intoxication, zinc deficiency, and magnesium deficiency.
For each patient, medical history was obtained using a standardized questionnaire indicating specified signs and symptoms as present or absent, and entering the history and date of use of specified medications.Data were collated by means of an electronic case report form.Patient-level data were de-identified by assigning project-specific codes (i.e., patient numbers) to the clinical data and biologic specimens.The protocol and an amendment were approved by the ethics committee of the medical faculty of the University of Rostock (Vote number A2020-0169) as well as by the ethics committee of the University of Würzburg (Vote number 221/20).The study was conducted in compliance with guidelines for Good Clinical Practice and the ethical principles of the Declaration of Helsinki.Written informed consent was obtained from patients prior to initiation of study procedures.

WGS
Kits containing the Centogene GmbH (Rostock, Germany) proprietary dry blood spot card (CentoCard) were used to collect blood samples from patients and were returned to Centogene for analysis.Genomic DNA was isolated from the CentoCard using a standard protocol developed by Centogene.After fragmentation of genomic DNA by sonication, Illumina adapters were ligated to generate fragments for subsequent sequencing on the HiSeqX platform (Illumina, Inc., San Diego, CA, USA) to yield an average coverage depth of more than 30X.

Clinical chemistry
Figure 1 shows mean (range) serum ALP activity for each patient measured on 2 occasions at least 1 month apart.Mean (SD; range) ALP enzyme activity was 30.81 (5.68; 17.90 − 40.0)U/L for the first measurement and 32.62 (5.80; 23.0 − 39.0) U/L for the second measurement.All measurements were below the normal age-and sex-adjusted range for ALP enzyme activity for adult males (53-128 U/L) and females (42-98 U/L) [14].

Genetic testing
All 16 patients received a genetic report and were confirmed by WGS to be negative for clinically relevant ALPL variants.Table 1 lists genes that were identified through OMIM search as regulators of ALPL or the HPP phenotype.Four patients (25%; patients 3, 4, 7, and 15) had positive WGS findings for variants in other genes that may interfere with ALP enzyme activity or result in some clinical manifestations mimicking HPP (Table 3).

Selection of target genes
Several databases, including the Online Mendelian Inheritance in Man (OMIM) database, the Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway Database, and PubMed, were used to identify a panel of candidate genes that may affect ALPL or the HPP phenotype.Specific search terms used to identify genes in peer-reviewed scientific papers and reviews published in the past 30 years included alkaline phosphatase activity, alkaline phosphatase, activity regulation, ALP activity, ALP activity regulation, ALPL regulation, HPP disease, and hypophosphatasia.Genes identified in the search of the OMIM database were added to the candidate gene list in the current study if pathogenic variants with perturbation of ALP were known and the mode of inheritance was specified.The KEGG Pathway Database was reviewed to identify upstream and downstream genes involved in ALP pathways; genes identified in this search were added to the list of candidate genes.From these candidates, a joint steering committee of investigators from Alexion, AstraZeneca Rare Disease and Centogene mutually agreed on a final panel of genes for analysis.Genes included in the analysis and their association with diseases similar to HPP are listed in Table 1.

Data analysis
An end-to-end in-house bioinformatics pipeline, including base calling, primary filtering of low-quality reads and probable artifacts, and annotation of variants was applied.Subsequently, all disease-causing variants reported in the Human Gene Mutation Database [10], ClinVar [11], or CentoMD, as well as variants with minor allele frequency below 1% in the Exome Aggregation Consortium (ExAC) database [12], were considered.In addition to considering all pertinent inheritance patterns, family history and clinical information were used to evaluate identified variants.All identified variants were assessed for their pathogenicity and causality and classified as pathogenic, likely pathogenic, VUS, likely benign, or benign according to the standards and guidelines of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology [13].All variants related to the patient's phenotype and CNVs of unknown significance were also reported.
To detect structural variants that might affect ALPL but may have been missed with exome sequencing, raw sequence data analysis was performed using the DRAGEN pipeline (Illumina, Inc.); this analysis included base calling, de-multiplexing, alignment to the hg19 human reference 1 3 collagen prolyl hydroxylase family implicated in autosomal recessive osteogenesis imperfecta [15].Testing outside the requested gene panel showed that Patient 7 (36-year-old woman) had a heterozygous pathogenic variant (NM_001346713.1:c.817C > T, leading to a premature stop codon) in SGCE, coding for sarcoglycan epsilon, a transmembrane protein that links the cytoskeleton protein, well as a CNV (heterozygous deletion) of SCN9A, coding for sodium voltage-gated channel alpha subunit 9, from chromosome 2 (genomic location, 167157718-167362541).
Patient 4 (43-year-old woman) was found to be a heterozygous carrier of a likely pathogenic variant (NM_001243246.1:c.1346-1G> A) in the P3H1 gene, which codes for prolyl 3-hydroxylase 1, a member of the ALP activity, or demonstrated some phenotypic overlap with HPP, we do not claim these to be causative of HPP.The mechanisms involved in this observed association remain unknown.Further research is needed to better understand the molecular basis of HPP in these ALPL variant-negative patients.
All 16 patients with clinical and laboratory results indicative of HPP were confirmed by WGS to be negative for pathogenic ALPL variants, including rare, non-coding variants with potential implications in gene splicing and gross copy number alterations.However, 4 of the 16 patients had findings for genetic variants in other genes with the potential for regulating ALP or modifying the HPP phenotype.Variants in COL1A1 are associated with bone disorders, including osteogenesis imperfecta types 1-4 [18].Osteogenesis imperfecta is a connective tissue disorder associated with bone fragility, making it a potential differential diagnosis of HPP [1,19].Variants in SCN9A are associated with primary erythromelalgia, paroxysmal extreme pain disorder, or insensitivity to pain [20], and variants in NLRP12 are associated with familial cold-induced autoinflammatory syndromes [21].Patient 3 reported history of pain and headache, which could correspond with these conditions but are nonspecific.Since this patient had no history of other, more specific manifestations or clinical presentations associated with these disorders, such as low bone mineral density, rash, redness, warmth, or swelling, the patient was not diagnosed with any of these disorders.
The P3H1 variant NM_001243246.1:c.1346-1G> A is a splice variant known to impair the function of the nearby acceptor splice site, and a variant at this position (with a different nucleotide exchange, c.1346-1G > C, ClinVar variation ID: 284532, clinical testing, listed as pathogenic) has been reported as disease-causing for autosomal recessive osteogenesis imperfecta type 8 [15].However, the patient's clinical phenotype was not consistent with osteogenesis imperfecta type 8 and a second relevant variant in P3H1 compatible with an autosomal-recessive trait was not demonstrable.Therefore, the patient is currently deemed to be an asymptomatic carrier of a pathogenic P3H1 variant.
The pathogenic variant in the SGCE gene (NM_001346713.1:c.817C > T) leads to a premature stop codon and has been described as pathogenic for MDS (OMIM: 159900), a rare movement disorder characterized by dystonia and lightning-like myoclonic jerks [22].As the SGCE locus is imprinted, approximately 95% of patients with MDS who inherit the variant from their mother will remain healthy, whereas almost all children who inherit the variant from their father will develop MDS [22].De novo variants are also described.Since the clinical picture of MDS has little overlap with that of the patient and cannot explain her symptoms (e.g., premature loss of permanent actin, to the extracellular matrix.This SGCE variant is involved in myoclonus dystonia syndrome (MDS) type 11, a disease with autosomal dominant inheritance that results in rapid, involuntary muscle jerks.No other variant relevant to the described phenotype was found.Patient 15 (51-yearold man) had a heterozygous VUS (positional mutation, NM_001017536.1:c.265A > G, [p.Met89Val]) in VDR, which codes for the vitamin D receptor.

Discussion
HPP is a disabling condition with wide-ranging impacts on health-related quality of life [16].However, accurate diagnosis and appropriate treatment are often delayed, owing, in part, to symptom overlap between HPP and more common conditions [1,17].Sequencing of the ALPL coding region confirms the diagnosis of HPP if an ALPL variant is identified; however, some patients with clinical and laboratory findings consistent with a diagnosis of HPP have negative genetic testing results [4].
The current study was performed to further elucidate the genetic underpinnings of ALPL and non-ALPL variants beyond what can be detected with standard sequencing of the ALPL coding region, including genes that may interfere with ALPL expression or processing, or activity of ALP, thus modifying the phenotypic presentation of HPP.
With regard to the primary objective of this study, WGS did not reveal any additional variants in ALPL that, according to the current knowledge, were considered pathogenetically relevant and caused reduced ALP activity and HPP symptoms in these patients.However, low ALP activity may be attributed to variation in the non-coding region of ALPL, since there is no definition of what constitutes normal ALPL intronic sequence vs. pathogenic variations in ALPL intronic sequences.
Results of this study with respect to the secondary objective of identifying novel genetic variants beyond ALPL that could be pathogenetically relevant do not provide an answer to the long-standing speculation that confounding genes modulate impact on either ALPL expression or ALP activity.Although we have observed variations in several genes that were a priori selected as potentially responsible for low VDR encodes the vitamin D receptor, a member of the nuclear hormone receptor superfamily of D-resistant rickets type 2A, a disease with an autosomal recessive inheritance [24].Vitamin D-resistant rickets type 2A or hypocalcemic vitamin D-resistant rickets is an inherited disorder of vitamin D characterized by hypocalcemia, severe rickets, and, in many cases, alopecia [24].Although not directly related to ALPL, variants in VDR may interact with ALPL at some level and influence the phenotypic presentation of the disease.The current study is not without limitations.The small study population was overrepresented by females from a single geographic region, reducing generalizability of the results.Accordingly, results of this analysis highlight the importance of future genomic studies in larger cohorts from a variety of geographic regions, including additional genes that may contribute to HPP manifestations, to better understand the genetic basis of HPP.In addition, future studies should investigate epigenetic and extragenomic factors via transcriptomic and metabolomics analyses.

Conclusions
The genetic basis of HPP in patients without ALPL variants remains unclear.In the present analysis, all patients were confirmed via WGS to be negative for variants in ALPL despite low ALP activity.Variants in non-ALPL genes did not conclusively overlap with genes associated with any other disorder and may contribute to the HPP phenotype through unknown mechanisms.These data provide an important rationale for further investigation of molecular mechanisms linking genes beyond ALPL to the HPP phenotype and of the genetic underpinnings of HPP.Nevertheless, diagnosis of HPP remains a clinical diagnosis based on signs and symptoms in affected individuals and does not require detection of ALPL variants [1].
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Table
Summary of positive results for genetic variants in genes whose products may regulate ALP activity or modify HPP phenotype Pt no.The protocol and an amendment were approved by the ethics committee of the medical faculty of the University of Rostock (Vote number A2020-0169) as well as by the ethics committee of the University of Würzburg (Vote number 221/20).The study was conducted in compliance with guidelines for Good Clinical Practice and the ethical principles of the Declaration of Helsinki.Written informed consent was obtained from patients prior to initiation of study procedures.Competing interests Lothar Seefried is a consultant for and has received research funding and honoraria from Alexion, AstraZeneca Rare Disease.Anna Petryk and Guillermo del Angel are employees of and may own stock/options in Alexion, AstraZeneca Rare Disease.Felix Reder is an employee of and may own stock/options in Centogene GmbH, which received funding from Alexion, AstraZeneca Rare Disease for the conduct of the study.Peter Bauer is a board member of and may own stock/options in Centogene GmbH.International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made.The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material.If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.To view a copy of this licence, visit http://creativecommons. org/licenses/by/4.0/.